RESUMO
The ability to cryopreserve organs would have an enormous impact in transplantation medicine. To investigate organ cryopreservation strategies, experiments are typically done on whole organs, or on cells in 2D culture. Whole organs are not amenable to high throughput investigation, while conventional 2D culture is limited to a single cell type and lacks the complexity of the whole organ. In this study, we examine kidney organoids as a model system for studying cryopreservation. Consistent with previous studies, we show that kidney organoids comprised of multiple cell types can be generated in 96-well plates, with an average of about 8 organoids per well. We present a live/dead staining and image analysis method for quantifying organoid viability and show that this method can be used for assessing cryoprotectant toxicity. Our results highlight the potential for using organoids for high throughput investigation of cryopreservation approaches.
Assuntos
Células-Tronco Pluripotentes Induzidas , Organoides , Criopreservação/métodos , Humanos , Rim , Modelos BiológicosRESUMO
Polycystic kidney disease (PKD) is a life-threatening disorder, commonly caused by defects in polycystin-1 (PC1) or polycystin-2 (PC2), in which tubular epithelia form fluid-filled cysts. A major barrier to understanding PKD is the absence of human cellular models that accurately and efficiently recapitulate cystogenesis. Previously, we have generated a genetic model of PKD using human pluripotent stem cells and derived kidney organoids. Here we show that systematic substitution of physical components can dramatically increase or decrease cyst formation, unveiling a critical role for microenvironment in PKD. Removal of adherent cues increases cystogenesis 10-fold, producing cysts phenotypically resembling PKD that expand massively to 1-centimetre diameters. Removal of stroma enables outgrowth of PKD cell lines, which exhibit defects in PC1 expression and collagen compaction. Cyclic adenosine monophosphate (cAMP), when added, induces cysts in both PKD organoids and controls. These biomaterials establish a highly efficient model of PKD cystogenesis that directly implicates the microenvironment at the earliest stages of the disease.
Assuntos
Microambiente Celular , Modelos Biológicos , Organoides/metabolismo , Doenças Renais Policísticas/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Humanos , Organoides/patologia , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/patologia , Canais de Cátion TRPP/biossíntese , Canais de Cátion TRPP/genéticaRESUMO
A critical event during kidney organogenesis is the differentiation of podocytes, specialized epithelial cells that filter blood plasma to form urine. Podocytes derived from human pluripotent stem cells (hPSC-podocytes) have recently been generated in nephron-like kidney organoids, but the developmental stage of these cells and their capacity to reveal disease mechanisms remains unclear. Here, we show that hPSC-podocytes phenocopy mammalian podocytes at the capillary loop stage (CLS), recapitulating key features of ultrastructure, gene expression, and mutant phenotype. hPSC-podocytes in vitro progressively establish junction-rich basal membranes (nephrin+ podocin+ ZO-1+ ) and microvillus-rich apical membranes (podocalyxin+ ), similar to CLS podocytes in vivo. Ultrastructural, biophysical, and transcriptomic analysis of podocalyxin-knockout hPSCs and derived podocytes, generated using CRISPR/Cas9, reveals defects in the assembly of microvilli and lateral spaces between developing podocytes, resulting in failed junctional migration. These defects are phenocopied in CLS glomeruli of podocalyxin-deficient mice, which cannot produce urine, thereby demonstrating that podocalyxin has a conserved and essential role in mammalian podocyte maturation. Defining the maturity of hPSC-podocytes and their capacity to reveal and recapitulate pathophysiological mechanisms establishes a powerful framework for studying human kidney disease and regeneration. Stem Cells 2017;35:2366-2378.
Assuntos
Organoides/metabolismo , Podócitos/metabolismo , Animais , Adesão Celular/genética , Adesão Celular/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Edição de Genes , Humanos , Rim/metabolismo , Rim/patologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismoRESUMO
In polycystic kidney disease (PKD), fluid-filled cysts arise from tubules in kidneys and other organs. Human kidney organoids can reconstitute PKD cystogenesis in a genetically specific way, but the mechanisms underlying cystogenesis remain elusive. Here we show that subjecting organoids to fluid shear stress in a PKD-on-a-chip microphysiological system promotes cyst expansion via an absorptive rather than a secretory pathway. A diffusive static condition partially substitutes for fluid flow, implicating volume and solute concentration as key mediators of this effect. Surprisingly, cyst-lining epithelia in organoids polarize outwards towards the media, arguing against a secretory mechanism. Rather, cyst formation is driven by glucose transport into lumens of outwards-facing epithelia, which can be blocked pharmacologically. In PKD mice, glucose is imported through cysts into the renal interstitium, which detaches from tubules to license expansion. Thus, absorption can mediate PKD cyst growth in human organoids, with implications for disease mechanism and potential for therapy development.
Assuntos
Cistos , Doenças Renais Policísticas , Humanos , Camundongos , Animais , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/metabolismo , Rim/metabolismo , Epitélio/metabolismo , Organoides/metabolismo , Cistos/metabolismoRESUMO
The plasma membrane permeability to water and cryoprotectant (CPA) significantly impacts vitrification efficiency of bovine oocytes. Our study was designed to determine the concentration-dependent permeability characteristics for immature (GV) and mature (MII) bovine oocytes in the presence of ethylene glycol (EG) and dimethyl sulphoxide (Me2SO), and to compare two different modeling approaches: the two parameter (2P) model and a nondilute transport model. Membrane permeability parameters were determined by consecutively exposing oocytes to increasing concentrations of Me2SO or EG. Higher water permeability was observed for MII oocytes than GV oocytes in the presence of both Me2SO and EG, and in all cases the water permeability was observed to decrease as CPA concentration increased. At high CPA concentrations, the CPA permeability was similar for Me2SO and EG, for both MII and GV oocytes, but at low concentrations the EG permeability of GV oocytes was substantially higher. Predictions of cell volume changes during CPA addition and removal indicate that accounting for the concentration dependence of permeability only has a modest effect, but there were substantial differences between the 2P model and the nondilute model during CPA removal, which may have implications for design of improved methods for bovine oocyte vitrification.
Assuntos
Criopreservação , Dimetil Sulfóxido/farmacologia , Etilenoglicol/farmacologia , Oócitos/efeitos dos fármacos , Animais , Bovinos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Crioprotetores/farmacologia , Oócitos/crescimento & desenvolvimentoRESUMO
Organoids derived from human pluripotent stem cells are a potentially powerful tool for high-throughput screening (HTS), but the complexity of organoid cultures poses a significant challenge for miniaturization and automation. Here, we present a fully automated, HTS-compatible platform for enhanced differentiation and phenotyping of human kidney organoids. The entire 21-day protocol, from plating to differentiation to analysis, can be performed automatically by liquid-handling robots, or alternatively by manual pipetting. High-content imaging analysis reveals both dose-dependent and threshold effects during organoid differentiation. Immunofluorescence and single-cell RNA sequencing identify previously undetected parietal, interstitial, and partially differentiated compartments within organoids and define conditions that greatly expand the vascular endothelium. Chemical modulation of toxicity and disease phenotypes can be quantified for safety and efficacy prediction. Screening in gene-edited organoids in this system reveals an unexpected role for myosin in polycystic kidney disease. Organoids in HTS formats thus establish an attractive platform for multidimensional phenotypic screening.